System For Detecting Infectious Agents Using Computer-Controlled Automated Image Analysis

ABSTRACT

A method for providing quantitative information regarding the extent of infection of host cells by an infectious agent. A microscope image of a specimen of a bodily fluid is analyzed using image processing techniques to quantify the percentage of the area of the specimen that is infected.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation application of U.S. patent application Ser. No.13/188,714, filed Jul. 22, 2011, which is a continuation application ofU.S. patent application Ser. No. 13/022,348, filed Feb. 7, 2011, whichis a continuation of U.S. patent application Ser. No. 12/052,898 filedMar. 21, 2008, now U.S. Pat. No. 7,885,449, which is a continuation ofU.S. patent application Ser. No. 10/543,270, filed Jun. 2, 2006, whichis a National Stage of International PCT Application NumberPCT/US02/17586 filed on Jun. 4, 2002 and published as WO02/098280 onDec. 12, 2002, which claims priority to U.S. Provisional PatentApplication No. 60/295,587 filed Jun. 4, 2001, each of which isincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to computer controlled methods andapparatus for detecting or detecting and quantifying an infectious agentin a biological sample. The identifying and/or quantitative dataobtained are useful in making a diagnosis or prognosis of diseases,including diseases classically deemed “non-infectious.” in one aspect,the invention relates to computer controlled methods and systems foridentifying an infected host, animal or plant cell in a field of cellsand thereby providing information useful in making a disease diagnosisor a prognosis of disease susceptibility based on identification of aninfected animal cell. In another aspect, the invention relates tocomputer controlled methods and systems for quantification of infectedhost, e.g., animal or plant cell(s) in a biological sample, therebyproviding information useful in making a disease diagnosis or aprognosis of disease susceptibility based on the quantification ofinfected cell(s). The quantification may be determination of the numberof infected animal cells, or of the extent to which the cells areinfected (i.e., the number of infectious agents in an infected cell) ordetermination of both numbers. The quantitative information also isuseful to assess therapeutic efficacy of treatment of disease.

In one important embodiment, the invention relates to quantification ofChlamydia infected mononuclear phagocyte(s) in a blood or tissue sampleto provide information useful to make a diagnosis or prognosis ofsusceptibility of vascular or coronary disease.

In another important embodiment, the invention relates to quantificationof Chlamydia infected mononuclear phagocyte(s) in a biological sample toprovide information helpful for or useful to make a diagnosis (orprognosis) of a central nervous system (CNS) disease or disorder.

In yet another aspect, the present invention relates to computercontrolled methods and systems for detecting and quantifying aninfectious agent “free floating” in a biological sample to provide datauseful in making a diagnosis or prognosis of disease or disorders,including diseases classically deemed “non-infectious”.

BACKGROUND OF THE INVENTION

Citation or identification of any reference in this section or anysection of this application shall not be construed as an admission thatsuch reference is available as prior art to the present invention.

Atherosclerosis is the main underlying cause of coronary heart diseaseand is characterized by the deposit of lipid containing plaques onendothelium of large and medium sized arteries. Atherosclerosis isthought to be initiated at dysfunctional vascular endothelium whennormal laminar blood flow is disrupted. Many systemic and local factorsmay cause dysfunctional endothelium and lead to or trigger aninflammatory response in the vessel wall. Multiple cell types canmediate this process, including monocyte-derived macrophages. Drexler,H. 1997. Prog. Cardiovasc. Dis. 39:287. The dysfunctional endotheliumallows passage of low density lipoprotein (“LDL”) cholesterol andexpresses multiple adhesion molecules for platelets and inflammatorycells. The LDL cholesterol undergoes partial oxidation and causesfurther endothelial dysfunction while monocytes penetrate theendothelium, differentiate into macrophages, and take up oxidized LDLcholesterol. The resulting lipid-laden macrophages, also known as foamcells, accumulate in the atherosclerotic lesion and ultimately mayrupture to release oxidized LDL cholesterol and cytotoxic enzymes. Thistriggers fibroproliferative responses from vascular smooth muscle cellsand leads to the development of atherosclerotic plaques. Fuster et al.1992. N. Engl. J. Med. 326:310; Stary, H. C. 1989. Arteriosclerosis99:1-19.

Studies have shown Herpes viruses, such as Herpes simplex virus andCytomegalovirus (“CMV”), can increase the risk of developing heartdisease. Roivainen et al. 2000, Circulation 101(3):252. For example,elevated CMV antibody titres are associated with the presence ofatherosclerosis. Melnick et al. 1990. JAMA 263:2204; Danesh et al. 1997,Lancet 350:430; Cheng et al. 2000, Expert Opin. Investig. Drugs9(11):2505. Based on pathological data demonstrating CMV DNA sequencesand viral inclusions in atherosclerotic lesions, 75 consecutive patientsundergoing directional coronary atherectomy for coronary disease werestudied to see if a link between CMV infection and arterial diseaseexists. The results showed that patients who were seropositive for CMVprior to the procedure have a greater than five-fold increased ratearterial disease. Zhou et al. 1996. N. Engl. J. Med. 335:624.

A mechanism by which CMV may affect atherosclerosis hinges on themononuclear phagocyte. CMV integrates into mononuclear cell precursorDNA thereby causing circulating monocytes to be a vector for deliveringvirus to sites of vessel inflammation. Guetta et al. 1997. Circ. Res.81:8. Macrophages have been shown to be a similar source of circulatingHUV in patients with AIDS. Orenstein et al. 1997. Science 276:1857.Studies have demonstrated that endothelial cells, smooth muscle cells,and oxidized LDL cholesterol can activate CMV viral replication ininfected mononuclear phagocytes which can lead to macrophage,endothelial cell, and vascular smooth muscle cell infection with CMV.CMV infected smooth muscle cells may then obtain growth advantages andcontribute to proliferative responses in atherosclerosis due to CMVinduced changes in expression of regulatory proteins. Speir et al. 1994.Science. 265:391.

A link between atherosclerosis and Helicobacter pylori has also beenshown. H. pylori is a Gram-negative rod which has been implicated in thedevelopment of peptic ulcers, gastric carcinoma, and low-grade B celllymphomas of the gastrointestinal tract. Schussheim et al. 1999, Drugs57:283. An association of H. pylori infection with coronary disease hasbeen suggested in which seropositivity conferred a two-fold increasedrisk of coronary artery disease among nearly 200 men. Mendall et al.1994. Br. Heart J. 71:437; Danesh et al. 1997, Lancet 350:430; Cheng etal. 2000, Expert Opin. Investig. Drugs 9(11):2505; Muhlestein, J. B.2000, Curr. Interv. Cardiol. Rep. 2(4):342. Another study supports thisassociation when it was seen that elevated serum fibrinogen levels andtotal leucocyte count were found more often in those seropositive for H.pylori. Patel et al. 1995. B.M.J. 311:711.

A link between Chlamydia pneumoniae and vascular disease, such asatherosclerosis and coronary disease or coronary syndrome is alsorecognized. Schussheim et al., 1999, Drugs 57:283; Roivainen et al.2000, Circulation 101(3):252; Muhlestein Curr. Interv. Cardiol. Rep.2(4):342; Danesh et al. 1997, Lancet 350:430; Cheng et al. 2000, ExpertOpin. Investig. Drugs 9(11):2505; Muhlestein, J. B. 2000, Curr. Interv.Cardiol. Rep. 2(4):342. Although Chlamydia is able to infect a number ofcell types, the bacteria's ability to infect mononuclear phagocytes isthought to be pivotal to its role in the development or modulation ofvascular disease, especially atherosclerosis. Mononuclear phagocytes arethought to spread infection from the respiratory tract to other organsystems based upon Chlamydia to remain metabolically active for at least10 days in mononuclear phagocytes infected in vitro. Moazed et al. 1998.J. Infect Des. 177:1322. Chlamydia can also stimulate the secretion ofproinflammatory cytokines such as tumor necrosis factor-.alpha.,interleukin [IL]-1 and interferon-.gamma. from monocytes and T cells.Saikku, P. 1997. J. Infect. Dis. 104:53; Kol et al. 1998. Circulation98:300; Hahne, S. 1997. Scand. J. Immunol. 45:378.

Several seroepidemiological studies now associate Chlamydia infectionwith atherosclerosis and promote the organism as a major pathologicalfactor of this general disease process. Saikku, P. 1997. Scand. J.Infect. Dis. 104:53; Campbell et al. 1998. Emerg. Infect. Dis. 4:571.Recently, C. pneumoniae-reactive T lymphocytes have been detected inhuman atherosclerotic plaques of the carotid artery. Mosorin, M., 2000,Arterioscler. Thromb. Vase. Biol. 20:1061. The authors of this studysuggest that Chlamydia, which is commonly detected in atheroscleroticplaque of the carotid and coronary arteries, causes T-cell activationand accumulation and this contributes to the maintenance of theinflammatory reaction in artherogenesis.

At least one epidemiological study has found that Chlamydia may bepresent as an associated agent in neurological infections. See,Koskiniemi, M. et al., 1996, Europ. Neurol., 36(3):60-63. A human.

SUMMARY

In its most general aspect, the present invention provides acomputer-implemented method of detecting at least one signal, whichprovides information which has diagnostic or prognostic significance.

In its most general embodiment, the method of the present inventionincludes acquiring image data of a sample of cells or a body fluid,processing the image data to select and record images of a detectablesignal indicative of an infectious agent. Counts may be maintained ofthe number and/or strength of the detectable signal identified. Theinfectious agent may be contained within the image of an animal cell orfree floating in the body fluid. Such counts provide for calculation ofthe extent of infection with an infectious agent(s).

In an embodiment, the image data is transformed from one color space,RGB (Red Green Blue) image into another color space, e.g., HLS (HueLuminescence Saturation) image. Filters and/or masks are utilized todistinguish those cells that meet pre-selected criteria, i.e. contain adetectable signal, and eliminate those that do not, and thus identifyinfected animal cells.

According to one embodiment, an infectious agent is detected orpreferably detected and quantified by computer controlled image analysisof host cells, e.g., animal or plant cells. In a preferred embodiment,the host cells are animal cells in a sample of a body fluid or tissuefrom an animal. Using this preferred method, animal cells infected withan infectious agent are detected or preferably detected and quantified.In the discussion below, animal cells are discussed as an exemplary“host” cell. As would be understood by those skilled in the art, this isfor discussion purposes only and the method would be understood to beuseful, in context, for detection of an infectious agent in any “host”,i.e., animal or plant cell.

According to this preferred embodiment of the method of the invention,computer controlled image analysis is conducted on a sample of bodyfluid or tissue containing animal cells in a monolayer treated toprovide at least two different signals which can both be detected andquantified. At least two signals are required. A first signal isemployed to identify an animal cell of interest and a second signal isemployed to identify an infectious agent within an identified animalcell of interest. Detection and quantification of the two differentsignals provides for determination of: (1) the number of infected animalcells, e.g., per unit volume of body fluid, (2) the number of infectiousagents per animal cell or (3) both the number of infected animal cellsand the number of infectious agents per cell, i.e., the extent of cellinfection.

According to the method, a monolayer of animal cells, fixed to asuitable solid substrate is observed by a computer controlled microscopesystem as described above. The monolayer can be obtained merely byspreading a body fluid or tissue sample with animal cells of interest ona solid substrate, such as a slide. Alternatively, a monolayer can beobtained by spreading a sample containing an enriched population ofanimal cells of interest on a solid substrate.

A physical feature of the animal cells can be used to provide a firstsignal, or more preferably the animal cells are stained to produce afirst signal. The fixed animal cells are also treated to produce asecond signal specific to an infectious agent, if said agent is present.

The computer controlled image analysis of this embodiment of theinvention is accomplished using a computer software product including acomputer-readable storage medium having fixed therein a sequence ofinstructions which, when executed by a computer directs the performanceof method steps comprising:

A microscope image of an optical field of a substrate having fixedthereon a monolayer of animal cells treated to produce a first signalspecific to a desired animal cell and a second signal specific to adesired infectious agent is acquired and transferred to the computer asan RGB image.

The Red component of the RGB image is transferred to a new monochromegrey-level image and clipped for pixel values of less than 50 to cutdown signal noise.

The grey-level image is transformed to a binary image, a black and whiteimage in which pixels with corresponding pixels in the Luminance imagehaving grey-level values lower than the cut off point are set to a valueindicative of the expected size of animal cells of interest (white).

An opening filter, successive applications of an erosion filter followedby a dilation filter, is applied for the removal of small noiseparticles from the binary image.

Application of a hole filling function fills the holes in the identifiedimages.

The area of each image is measured and all images having an area ofpixels equal to or greater than the expected value of the animal cell ofinterest are selected as representative of animal cells. Cell imagesthat have an area less than said pixels are excluded from furtherprocessing.

The area, in pixels, of the cell images, i.e., animal cells, is recordedand saved for farther processing. In one embodiment, in which all cellimages are to be quantified in a sample, the XY location of each cellimage is recorded.

The Red component from the original RGB image is transferred to a binaryimage, so that pixels having grey-level values less than the expectedvalue of the signal indicative of an infectious agent are set to 0 whileall the rest are set to 255.

The Green component from the original RGB image is transferred to greylevel image.

All pixels that have a value equal to a set value M and any grey-levelvalue in the Green component of the original RGB image equal to a setvalue N, together indicative of a particular signal form a newgrey-level image.

This new grey-level image is transformed into a binary image wherepixels that have grey-level values less than 100 are set to 0 and therest of the pixels are set to.

An opening filter is applied to remove small noise particles from thebinary image and a hole filling function is applied.

The total area of the remaining cell images, which represents the totalarea of infectious agent within the cell perimeter, is recorded.

Percent area of each animal cell that is occupied by an infectious agentcan be calculated.

According to one mode of this embodiment of the invention, detection ofinfected animal cells can simply be recorded for each image or theprocess can be repeated for a number of optical fields and a number ofimages can be accessed up to an including the entire surface of a slide.Simple detection of infected animal cells provides useful informationfor diagnosis and/or prognosis as described in detail in Section 5.2,infra. In addition, the number of infected animal cells can bedetermined to provide even more useful information as described inSection 5.2, infra.

Alternatively, the percent area of an animal cell occupied by aninfectious agent can be calculated. This provides quantitativeinformation regarding extent of infection useful for diagnosis and/orprognosis as described in detail in Section 5.2, infra.

Additionally, according to an alternative of this mode of the embodimentof the invention, the number and positions of all animal cells on aslide can be determined.

According to another embodiment, an infectious agent is detected orpreferably detected and quantified by computer controlled image analysisof a free floating infectious agent in a sample of a body fluid ortissue from an animal. In this embodiment, computer controlled imageanalysis is conducted on a sample of body fluid or tissue treated toprovide at least one signal specific to the infectious agent of interestwhich can be detected and quantified.

The computer controlled image analysis of this embodiment of theinvention is accomplished using a computer software product including acomputer-readable storage medium having fixed therein a sequence ofinstructions which, when executed by a computer directs the performanceof method steps comprising:

A microscope image of an optical field of a substrate having fixedthereon a sample of a body fluid or tissue treated to produce a signalspecific to a desired infectious agent is acquired and transferred tothe computer as an RGB image.

The image is transferred to the HLS domain.

The Hue component of the HLS image is transformed to a new monochromegreylevel image.

The greylevel image is transformed to a “binary” image: this is a blackand white image in which pixels with pixels having values between 10 and25 in the Hue image are set to 255 (white) and the rest being set to 0black.

An “open” filter is applied: Opening is a successive application of an“erosion” filter followed by a “dilation” filter. It allows for theremoval of small noise particles from the binary image and a holefilling function is applied that “fills the holes” in the identifiedblobs.

The area of the blobs is measured and all blobs that have an area ofless than the expected area or size of the infectious agent are excludedfrom further processing. In an illustrative example, when the infectiousagent is Chlamydia, all blobs that have an area of less than 30 pixelsand more than 70 pixels are excluded from further processing. Theremaining blobs represent infectious agent in the blood sample.

The pixel number (area) of these blobs (cells) is recorded and saved forfurther processing.

The information provided by the computer controlled image analysismethods of the present invention is useful, preferably in combinationwith other information relating to the physical and/or physiologicalstate of a human or non-human patient in diagnosis of prognosis of adisease or disorder associated with infection of the human or non-humanpatient by an infectious agent. The information provided is useful fordiagnosis or prognosis of diseases and disorders associated with avariety of infectious agents, including, but not limited to, prions,bacteria, mycoplasma, rickettsia, spirochetes, fungi, protozoalparasites, viruses, etc.

One particular embodiment of the present invention is acomputer-implemented method for detecting a Chlamydia infectedmononuclear phagocytes from a blood or tissue sample.

Another particular embodiment of the present invention is acomputer-implemented method for detecting Helicobacter pylori infectedmononuclear phagocytes from a blood or tissue sample.

Another particular embodiment of the present invention is acomputer-implemented method for detecting CMV infected mononuclearphagocytes from a blood or tissue sample.

Another particular embodiment of the present invention is acomputer-implemented method for detecting Herpes simplex virus infectedmononuclear phagocytes from a blood or tissue sample.

Another particular embodiment of the present invention is acomputer-implemented method for detecting mononuclear phagocytesinfected with one or more of a variety of periodontal infectious agentssuch as P. gingivalis, S. sanguis, from a blood or tissue sample.

The present invention also encompasses a computer software productincluding a computer-readable storage medium having fixed therein asequence of instructions which, when executed by a computer, direct theperformance of steps for conducting the methods of the invention asdescribed herein.

The present invention also encompasses to a method of operating alaboratory service for providing useful information for diseasediagnosis and prognosis of disease extent or susceptibility or efficacyof a therapeutic treatment. The method encompasses the steps ofreceiving a prepared substrate, e.g. a slide, that has a monolayer ofcells from a body fluid or tissue sample or receiving a body fluid ortissue sample where the body fluid or tissue sample is placed on asubstrate as a monolayer, treating the sample to generate adiagnostic/prognostic signal, obtaining an image of a monolayer ofcells, and operating a computerized microscope according to themethod(s) described herein to provide the information useful to diagnosea disease or prognosticate disease susceptibility.

The present invention also provides a system for screening infectedanimal cells or infected body fluids or tissues. The basic elements ofthe system include an X-Y stage, a mercury light source, a fluorescencemicroscope, digital camera system such as a color CCD camera, or acomplementary metal-oxide semiconductor (CMOS) image system, a personalcomputer (PC) system, and one or two monitors and most importantly acomputer software product including a computer-readable storage mediumhaving fixed therein a sequence of instructions which, when executed bya computer, direct the performance of steps for conducting the methodsof the invention as described herein. In a preferred embodiment, thestage has an automated microscope feeder configured with it so thatslides can be automatically moved into and out of position for imagecapture and/or analysis.

3.1. OBJECTS AND ADVANTAGES OF THE INVENTION

It is an object of the invention to provide methods and systems whichprovide quantitative information regarding animal (human or non-human)cells infected with an infectious agent. Unlike other methods whichprovide merely for the detection of an infectious agent, the presentmethods and systems advantageously provide quantitative information withrespect to infectious agents present in animal cells. Such information,preferably, in combination with other information relating to thephysical and/or physiological state of a human or non-human patient, isuseful in diagnosis or prognosis of a disease or disorder associatedwith the presence infectious agent. Unlike other methods which merelydetect the presence of an infectious agent, in certain embodiments themethods and systems of the present invention can provide detailedinformation relating to the life cycle state and form of an infectiousagent.

It is another object of the invention to provide methods and systemswhich provide quantitative information regarding the presence of aninfectious agent “free floating” in an animal human or non-human) bodyfluid or tissue sample. Such quantitative information, preferably incombination with other family/genetic heritage, genetic profile,physical and/or physiological information relating to the heredity,physical and/or physiological state of a human or non-human patient, isuseful in prediction of susceptibility, diagnosis or prognosis of adisease or disorder associated with the presence of the infectiousagent.

In certain embodiments, the quantitative information provided by themethods of the invention is used to assess therapeutic efficacy of atreatment which the human or non-human patient is undergoing.

In certain other embodiments, the quantitative information provided bythe methods of the invention is used to assess susceptibility of thehuman or non-human animal to recurrence or progress of a recurring orprogressive disease or disorder.

In certain other embodiments, the quantitative information provided bythe methods of the invention, along with information regarding thefamily history and/or genetic profile of relevant genes of a patient isused to predict chance of onset of a disease, advantageously prior toonset of clinical symptoms. In certain modes of these embodiments, theinformation is included in a “wellness” check or profile of the patient.For one example, application of the methods of the invention to monitorpatient samples “over” time can be used to assess a patient's ability toclear a bacterial infection from monocytes and hence assesssusceptibility to developing vascular disease.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention may be understood more fully by reference to thefollowing detailed description of the invention, illustrative examplesof specific embodiments of the invention and the appended figures inwhich:

FIG. 1 is schematic representation of an apparatus or system useful forthe methods of the invention. The automated microscope slide feeder(109) depicted in FIG. 1 is an optional component of a preferred systemuseful for the methods of the invention.

FIG. 2 is a flow chart summarizing the method of one aspect of theinvention in which an infectious agent is detected or detected andquantified in an animal cell, in FIG. 2, the infectious agent isexemplified by Chlamydia; however, as would be understood by thoseskilled in the art, any of the infectious agents taught in thisapplication could be detected and quantified according to the methodillustrated in FIG. 2.

FIG. 3 is a more detailed flow chart of portions of an alternativeembodiment of the method of the aspect of the invention shown in FIG. 2.

FIG. 4 is a flow chart summarizing the method of another aspect of theinvention in which an infectious agent is detected or detected andquantified “free floating” in a biological sample from an animal.

DETAILED DESCRIPTION OF INVENTION

The invention will be better understood upon reading the followingdetailed description of the invention and of various exemplaryembodiments of the invention, in connection with the accompanyingdrawings. It will be clear to those skilled in the art that theinvention can be applied to and, in fact, encompasses methods andsystems to provide information useful in making a disease diagnosis or aprognosis of disease or disease susceptibility or outcome based ondetection and preferably, quantification of any characteristic resultingfrom infection of a host, e.g., an animal cell with an infectious agent.The animal cell can be obtained from any fluid or tissue sample fromwhich it is possible to view a monolayer of cells infected with saidinfectious agent. It will also be clear to those skilled in the art thatthe invention can be applied to, and in fact, encompasses methods andsystems to provide information useful in making a disease diagnosis orprognosis of disease or disease susceptibility or outcome based ondetection, and preferably quantification, of any characteristicindicative of the presence of an infectious agent “free floating” in abody fluid or tissue sample.

For the purpose of this invention, information useful for diseasediagnosing or prognosis, evaluation of patient wellness or prediction ofdisease onset is based upon detection of a signal associated with acharacteristic resulting from an infection of a animal cell, tissue orbody fluid. The term “characteristic” as used herein is defined to meanany non-endogenous substance(s) that indicates the presence of aninfectious agent in a animal cell, issue or body fluid, e.g. DNA, RNA,protein, carbohydrate, endotoxin, etc. indicative of or specific to aninfectious agent. Fluid and tissue samples that can be used in themethods of the invention include but are not limited to blood or a“fraction” thereof such as white blood cells or a specific category ofwhite blood cells, etc., tissue biopsies, spinal fluid, meningeal fluid,urine, alveolar fluid, etc.

In one aspect, the invention is described in connection with observing a“monolayer” of cells. Monolayer has a specific meaning as used herein.It means simply that the cells are arranged whereby they are not viewedon top of one another. It does not require confluence and can involvesingle cell suspensions. Thus, a monolayer can be viewed in smears ofsingle cell suspensions or a layer of tissue that has a thickness of asingle cell. Any solid or exfoliative cytology technique may also beemployed. Cells may be dissociated by standard techniques known to thoseskilled in the art. These techniques include but are not limited totrypsin, collagenase or dispase treatment of the tissue.

In another aspect, the invention is described in connection withobserving a “free floating” infectious agent. Free floating has aspecific meaning as used herein. It means simply that the infectiousagent present in a body fluid or tissue is not contained within theconfines of an animal cell. It does not require that the infectiousagent “float” in a biological fluid or tissue and the infectious agentcan be attached e.g., to the periphery of a cell or tissue. It simply isnot inside an animal cell.

According to a preferred embodiment of the invention, the animal cellsare mononuclear phagocytes. As used herein, the term “mononuclearphagocytes” is intended to comprise, without limitation, monocytesobtained from a central or peripheral blood, macrophages obtained fromany site, including any tissue or cavity, macrophages derived byincubating or culturing macrophage precursors obtained from bone marrowor blood. According to a more preferred embodiment, the mononuclearphagocytes are obtained from peripheral blood samples.

For the purpose of this invention, an “infectious agent” is intended tomean an organism or other agent, capable of replication and associatedwith a disease or disorder when said agent infects a host, such as ananimal host or a plant host. Infectious agents include, but are notlimited to, prions, bacteria, mycoplasma, rickettsia, spirochetes,fungi, parasites, protozoa, viruses, etc.

In an important embodiment, the infectious agent is a Chlamydia species.Common Chlamydia include: C. pneumoniae, C. trachomatis, C. psittaci andC. pecorum.

In another important embodiment, the infectious agent is a Helicobacterspecies. Common Helicobacter include: H. pylori and H. felis.

In another important embodiment, the infectious agent is a Borreliaspecies. Common Borrelia include: B. burgdorferi and B. recurrentis.

In another important embodiment, the infectious agent is a commonperiodontal bacteria. Common periodontal bacteria include: P.gingivalis, and P. sanguis.

In yet another important embodiment, the infectious agent is a Herpesvirus, such as Herpes simplex virus or Cytomegalovirus.

Solely for ease of explanation, the description of the invention isdivided into the following sections: (1) methods and systems (includingapparatus) for detection and quantification of an infectious agent,including quantification of an infectious agent (a) in an animal celland (b) “free floating” in a body fluid or tissue sample: and (2)applications and diseases and disorders for which useful information isprovided.

5.1. METHODS AND SYSTEMS FOR DETECTION OR DETECTION AND QUANTIFICATION

The present invention provides methods, apparatus and systems for therapid detection and, more preferably, detection and quantification of aninfectious agent in a biological sample. The methods employ apparatusand systems which afford computer controlled automated image analysis.

FIG. 1 illustrates the basic elements of a system suitable for useaccording to the methods of the invention. A system such as illustratedin FIG. 1 can be used in any of die methods described in thesub-sections below. The basic elements of the system include an X-Ystage (101), a mercury light source (102), a fluorescence microscope(103), a color CCD camera or CMOS image sensor (105), a personalcomputer (PC) system (106), and one or two monitors (107 and 108). In apreferred embodiment, the stage has an automated microscope feeder (109)configured with it so that slides can be automatically moved into andout of position for image capture and/or analysis.

The individual elements of the system can be custom built or purchasedoff-the-shelf as standard components. Each element is described insomewhat greater detail below.

The X-Y stage can be any motorized positional stage suitable for usewith the selected microscope. Preferably, the X-Y stage can be amotorized stage that can be connected to a personal computer andelectronically controlled using specifically compiled software commands.When using such an electronically controlled X-Y stage, a stagecontroller circuit card plugged into an expansion bus of the PC connectsthe stage to the PC. The stage should also be capable of being drivenmanually. Electronically controlled stages such as described here areproduced by microscope manufacturers, for example including Olympus(Tokyo, Japan), as well as other manufacturers, such as LUDL (NY, USA).

The microscope can be any fluorescence microscope equipped with areflected light fluorescence illuminator and a motorized objective lensturret with a 20.times., 40.times., and 100.times. (with or without oilimmersion) objective lens, providing a maximum magnification of1000.times. The motorized nosepiece is preferably connected to the PCand electronically switched between successive magnifications usingspecifically compiled software commands. When using such anelectronically controlled motorized nosepiece, a nosepiece controllercircuit card plugged into an expansion bus of the PC connects the stageto the PC. The microscope and stage are set up to include a mercurylight source, capable of providing consistent and substantially evenillumination of the complete optical field.

The microscope produces an image viewed by the camera. The camera can beany color 3-chip CCD camera or other camera or digital image sensorsystem connected to provide an electronic output and providing highsensitivity and resolution. The output of the camera is fed to a framegrabber and image processor circuit board installed in the PC. A camerafound to be suitable is the OPTRONICS 750 (OPTRONICS, CA.). Anotherdigital camera found to be suitable is the complementary metal-oxidesemiconductor system (CMOS) image sensor available from Photobit(Pasadena, Calif.).

Various frame grabber systems can be used in connection with the presentinvention. The frame grabber can be, for example, the MATROX GENESISavailable from MATROX (Montreal, CANADA). The MATROX GENESIS modulefeatures on-board hardware supported image processing capabilities.These capabilities compliment the capabilities of the MATROX IMAGINGLIBRARY (MIL) software package. Thus, it provides extremely fastexecution of the MIL based software algorithms. The MATROX boardssupport display to a dedicated SVGA monitor. The dedicated monitor isprovided in addition to the monitor usually used with the PC system. Anymonitor SVGA monitor suitable for use with the MATROX image processingboards can be used. One dedicated monitor usable in connection with theinvention is a ViewSonic 4E (Walnut Creek, Calif.) SVGA monitor.

In order to have sufficient processing and storage capabilitiesavailable, the PC can be any PC such as an INTEL PENTIUM-based PC havingat least 256 MB RAM and at least 40 GB of hard disk drive storage space.The PC preferably further includes a monitor. Other than the specificfeatures described herein, the PC is conventional, and can includekeyboard, printer or other desired peripheral devices not shown.

Alternatively, automated sample analysis may be performed by anapparatus and system for distinguishing, in an optical field, objects ofinterest from other objects and background, such as the automated systemexemplified in U.S. Pat. No. 5,352,613, issued Oct. 4, 1994(incorporated herein by reference). Furthermore, once an object, i.e., arelevant animal cell has been identified, the color, e.g. thecombination of the red, green, blue components for the pixels thatcomprise the object, or other parameters of interest relative to thatobject, i.e., whether it is infected or not by an infectious agent, canbe measured and stored.

Other examples of alternative apparatus and systems for automated sampleanalysis are illustrated in WO99/58972 and PCT/US00/31494 (incorporatedherein by reference).

One suitable system consists of an automatic microscopical sampleinspection system having:

a sample storage module and loading and unloading module

a sample transporting mechanism to and from an automated stage thatmoves the sample under a microscope objective lenses array

an array of CCD cameras

a processing unit having a host computer, multiple controllers tocontrol all mechanical parts of the microscopy system and

a high speed image processing unit where the CCD cameras are connected(see, PCT/US00/31494).

An innovative feature of this embodiment of a computer controlled systemis an array of two or more objective lenses having the same opticalcharacteristics. The lenses are arranged in a row and each of them hasits own z-axis movement mechanism, so that they can be individuallyfocused. This system can be equipped with a suitable mechanism so thatthe multiple objective holder can be exchanged to suit the same varietyof magnification needs that a common single-lens microscope can cover.Usually the magnification range of light microscope objectives extendsfrom 1.times. to 100.times.

Each objective is connected to its own CCD camera. The camera field ofview characteristics are such that it acquires the full area of theoptical field as provided by the lens.

Each camera is connected to an image acquisition device. This isinstalled in a host computer. For each optical field acquired, thecomputer is recording its physical location on the microscopical sample.This is achieved through the use of a computer controlled x-y mechanicalstage. The image provided by the camera is digitized and stored in thehost computer memory. With the current system, each objective lens cansimultaneously provide an image to the computer, each of which comprisesa certain portion of the sample area. The lenses should be appropriatelycorrected for chromatic aberrations so that the image has stablequalitative characteristics all along its area.

The imaged areas will be in varying physical distance from each other.This distance is a function of the distance at which the lenses arearranged and depends on the physical dimensions of the lenses. It willalso depend on the lenses' characteristics, namely numerical apertureand magnification specifications, which affect the area of the opticalfield that can be acquired. Therefore, for lenses of varyingmagnification/numerical aperture, the physical location of the acquiredimage will also vary.

The computer will keep track of the features of the objectives-array inuse as well as the position of the motorized stage. The storedcharacteristics of each image can be used in fitting the image in itscorrect position in a virtual patchwork, e.g. “composed” image, in thecomputer memory.

The host computer system that is controlling the above configuration, isdriven by software system that controls all mechanical components of thesystem through suitable device drivers. The software also comprisesproperly designed image composition algorithms that compose thedigitized image in the computer memory and supply the composed image forprocessing to further algorithms. Through image decomposition, synthesisand image processing specific features particular to the specific sampleare detected.

5.1.1. METHODS FOR DETECTION OR DETECTION AND QUANTIFICATION OF AGENTINSIDE AN ANIMAL CELL

According to one embodiment, an infectious agent is detected orpreferably detected and quantified by computer controlled image analysisof animal cells in a sample of a body fluid or tissue from an animal.Using this method, animal cells infected with an infectious agent aredetected or preferably detected and quantified.

According to this embodiment of the method of the invention, computercontrolled image analysis is conducted on a sample of body fluid ortissue containing animal cells in a monolayer treated to provide atleast two different signals which can both be detected and quantified.At least two signals are required. A first signal is employed toidentify an animal cell of interest and a second signal is employed toidentify an infectious agent within an identified animal cell ofinterest. Detection and quantification of the two different signalsprovides for determination of: (1) the number of infected animal cells,e.g., per unit volume of body fluid, (2) the number of infectious agentsper animal cell or (3) both the number of infected animal cells and thenumber of infectious agents per cell, i.e., the extent of cellinfection.

As used herein, “signal” should be taken in its broadest sense, as aphysical manifestation which can be detected and identified, thuscarrying information. One simple and useful signal is the light emittedby a fluorescent dye selectively bound to a structure of interest. Thatsignal indicates the presence of the structure, i.e., either an animalcell or an infectious agent, which might be difficult to detect absentthe fluorescent dye.

The requirements and constraints on the generation of the first signaland second signal are relatively simple. The materials and techniquesused to generate the first signal should not interfere adversely withthe materials and techniques used to generate the second signal (to anextent which compromises unacceptably detection and quantification), andvisa versa. Nor should they damage or alter the cell- or infectiousagent-specific characteristics sought to be measured to an extent thatcompromises unacceptably the detection and quantification. Finally, anyother desirable or required treatment of the cells should also notinterfere with the materials or techniques used to generate the firstsignal and second signal to an extent that compromises unacceptably thedetection and quantification. Within those limits, any suitablegenerators of the first signal and second signal may be used.

According to the method, a monolayer of animal cells, fixed to asuitable solid substrate is observed by a computer controlled microscopesystem as described above. The monolayer can be obtained merely byspreading a body fluid or tissue sample with animal cells of interest ona solid substrate, such as a slide. Alternatively, a monolayer can beobtained by spreading a sample containing an enriched population ofanimal cells of interest on a solid substrate. Enrichment of animalcells of interest can be accomplished by any means known to thoseskilled in the art, such as centrifugation, affinity purification, etc.A physical feature of the animal cells can be used to provide a firstsignal, or more preferably the animal cells are stained to produce afirst signal. The fixed animal cells are also treated to produce asecond signal specific to an infectious agent, if said agent is present.Optionally, the method is conducted using animal cells already fixed ona substrate, e.g. a slide. Alternatively, as part of the method of thepresent invention, the cells may be prepared on a substrate before beingtreated. Preparing the cells may be accomplished by first obtaining asample, washing the cells, and fixing the cells on a substrate. Itshould be evident that any means of obtaining, collecting, washing, andfixing the cells may be used depending on the infected animal cell to bedetected.

The body fluid or tissue sample may be obtained by a number of waysdepending upon where the infected cell resides. If, for example, theinfected cell resides in blood, a minimally invasive procedure, such astaking a blood sample, may be all that is necessary to obtain the cells.As an illustrative example, Chlamydia infected mononuclear phagocytescan be observed and quantitated in a peripheral blood sample. A similarminimally invasive procedure may be used if the infected cell resides ina mucosa layer. In an alternative embodiment of the present invention, asample may be taken by a more invasive procedure if desired. Forexample, a biopsy may be performed in order to obtain a sample toidentify an infected animal cell needed to make a diagnosis or prognosiswith the present invention.

Treating the cells may occur with a fluorescent dye bound to an antibodyagainst the characteristic that specifically identifies the infectiousagent. The animal cells may be simply counterstained with an agent thatfluoresces at a wavelength different from that of the fluorescent dyebound to the antibody specific for the infectious agent. Alternatively,animal cells are treated with a fluorescent dye bound to an antibodyspecific for the animal cells which dye fluoresces at a wavelengthdifferent from that of the dye bound to the antibody specific for theinfectious agent. It should now be evident that any two detectableindicators of the presence of an infected animal cell may serve as thefirst and second signals, subject to certain constraints noted herein.

As would be understood by those skilled in the art, for each infectiousagent to be detected, specific reagents are required to generate thesignals needed to detect the infected animal cell. For example, for theembodiment in which Chlamydia infected mononuclear phagocytes aredetected, a labelled antibody directed to Chlamydia can be used ingenerating the signal. Three examples of commercially availableantibodies are: (1) ChlamydiaCel, a genus-specific lipopolysaccharidemonoclonal antibody that identifies C. pneumoniae, as well as C.trachomatis and C. psittaci, with a strong fluorescence of theintracellular inclusions, the pinhead-sized elementary bodies and thefree cell-associated Chlamydia LPS antigens (CelLabs, Sydney,Australia), (2) Chlamydia CelPn, a C. pneumoniae specific monoclonalantibody (CelLabs, Sydney, Australia), and (3) a Chlamydia specificFITC-conjugated monoclonal antibody (Kallestad Diagnostics, Chaska,Minn.). Other illustrative examples of commercially available antibodiesspecific to cells of interest or infectious agents of interest that areuseful in the methods and systems of the invention include: Clone MCA38,mouse monoclonal antibody to human monocytes/macrophages (ResearchDiagnostics Inc., Flanders, N.J.); Clone B2, monoclonal antibody for thedetection of cytomegalovirus (Research Diagnostics Inc., Flanders,N.J.); Clone A33, monoclonal antibody for the detection of herpessimplex virus (Research Diagnostics Inc., Flanders, N.J.); Antibody05-97-92, affinity purified antibody to Borrelia species (Kirkegaard andPerry Laboratories, Gaithersburg, Md.); and Monoclonal antibodyA94000136P, to Helicobacter pylori, (BiosPacific, Emeryville, Calif.).

As would be understood by those skilled in the art, antibodies orantibody fragments specific for any host cell of interest or anyinfectious agent can be produced by any method known in the art for thesynthesis of antibodies (or binding fragments thereof, in particular, bychemical synthesis or preferably, by recombinant expression techniques.

Polyclonal antibodies to a host cell of interest or in infectious agentof interest can be produced by various procedures well known in the art.For example, a host cell of interest, an infectious agent or antigenthereof can be administered to various host animals including, but notlimited to, rabbits, mice, rats, etc. to induce the production of seracontaining polyclonal antibodies specific for the infectious agent orhost cell or antigen thereof. Various adjuvants may be used to increasethe immunological response, depending on the host species, and includebut are not limited to, Freund's (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. Briefly,mice can be immunized with a host cell of interest or antigenic partthereof, infectious agent or antigen thereof and once an immune responseis detected, e.g., antibodies specific for the host cell of interest orinfectious agent are detected in the mouse serum, the mouse spleen isharvested and splenocytes isolated. The splenocytes are then fused bywell known techniques to any suitable myeloma cells, for example cellsfrom cell line SP20 available from the ATCC. Hybridomas are selected andcloned by limited dilution. The hybridoma clones are then assayed bymethods known in the art for cells that secrete antibodies capable ofbinding an infectious agent or host cell of interest. Ascites fluid,which generally contains high levels of antibodies, can be generated byimmunizing mice with positive hybridoma clones.

Antibody fragments which recognize specific epitopes of a host cell orof an infectious agent may be generated by any technique known to thoseof skill in the art. For example, Fab and F(ab′)2 fragments may beproduced by proteolytic cleavage of immunoglobulin molecules, usingenzymes such as papain (to produce Fab fragments) or pepsin (to produceF(ab′)2 fragments). F(ab′)2 fragments contain the variable region, thelight chain constant region and the CHI domain of the heavy chain.Further, the antibodies useful to generate a signal (e.g., whenlabelled) can also be generated using various phage display methodsknown in the art.

In phage display methods, functional antibody domains are displayed onthe surface of phage particles which carry the polynucleotide sequencesencoding them. In particular, DNA sequences encoding VH and VL domainsare amplified from animal cDNA libraries (e.g., human or murine cDNAlibraries of lymphoid tissues). The DNA encoding the VH and VL domainsare recombined together with an scFv linker by PCR and cloned into aphagemid vector (e.g., p CANTAB 6 or pComb 3 HSS). The vector iselectroporated in E. coli and the E. coli is infected with helper phage.Phage used in these methods are typically filamentous phage including fdand M13 and the VH and VL domains are usually recombinantly fused toeither the phage gene III or gene VIII. Phage expressing an antigenbinding domain that binds to a host cell or an infectious agent ofinterest can be selected or identified with a host cell infectious agentor antigen thereof, e.g., using labeled antigen or antigen bound orcaptured to a solid surface or bead. Examples of phage display methodsthat can be used to make the antibodies useful in the method of thepresent invention include those disclosed in Brinkman et al., 1995, J.Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods184:177-186; Kettleborough et al., 1994, Eur. J. Immunol. 24:952-958;Persic et al., 1997, Gene 187:9-18; Burton et al., 1994, Advances inImmunology 57:191-280: PCT application No. PCT/GB91/O1 134; PCTpublication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO93/1 1236, WO 95/15982, WO 95/20401, and WO97/13844; and U.S. Pat. Nos.5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753,5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727,5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies or any other desired antigen binding fragment,and expressed in any desired host, including mammalian cells, insectcells, plant cells, yeast, and bacteria, e.g., as described below.Techniques to recombinantly produce Fab, Fab′ and F(ab′)₂ fragments canalso be employed using methods known in the art such as those disclosedin PCT publication No. WO 92/22324; Mullinax et al., 1992, BioTechniques12(6):864-869; Sawai et al., 1995, AJRI 34:26-34; and Better et al.,1988, Science 240:1041-1043 (said references incorporated by referencein their entireties).

To generate whole antibodies, PCR primers including VH or VL nucleotidesequences, a restriction site, and a flanking sequence to protect therestriction site can be used to amplify the VH or VL sequences in scFvclones. Utilizing cloning techniques known to those of skill in the art,the PCR amplified VH domains can be cloned into vectors expressing a VHconstant region, e.g., the human gamma 4 constant region, and the PCRamplified VL domains can be cloned into vectors expressing a VL constantregion, e.g., human kappa or lamba constant regions. Preferably, thevectors for expressing the VII or VL domains comprise an EF-1.alpha.promoter, a secretion signal, a cloning site for the variable domain,constant domains, and a selection marker such as neomycin. The VH and VLdomains may also cloned into one vector expressing the necessaryconstant regions. The heavy chain conversion vectors and light chainconversion vectors are then co-transfected into cell lines to generatestable or transient cell lines that express full-length antibodies,e.g., IgG, using techniques known to those of skill in the art.

Alternatively, the second signal, which indicates the presence of aparticular characteristic of an infectious agent being tested for, andmay be generated, for example, using technologies in which nucleic acidsspecific to the infectious agent are labelled and detected usingtechniques including such as in situ PCR-amplification or PCR in situhybridization or FISH.

Cells that emit both signals, e.g. the cell is an infected animal celland contains the characteristic being tested for with the second signal,are scored. Counts may be maintained of the number and strengths of thefirst signal and second signal detected.

While the description herein explains the method with respect tomononuclear phagocytes (or monocytes) as the host, e.g., animal cells;blood as the body fluid or tissue sample and Chlamydia as the infectiousagent, it would be understood by those skilled in the art that theinvention can be applied to and in fact, encompasses computer controlledautomated image analysis of any type of host cell and any body fluid ortissue sample infected by any relevant infectious agent. Hence, it isunderstood that the following description is merely explained, forpurposes of ease of explanation and is illustrated in FIGS. 2 and 3,with respect to detection and quantification of mononuclear phagocytesinfected with Chlamydia.

FIG. 2 schematically illustrates one embodiment of one method of theinvention, using monocytes as the animal cells and Chlamydia as theinfectious agent, merely for ease of explanation.

An objective, e.g., the 100.times. objective is selected to providetotal magnification of 1000.times. and a slide containing a monolayer oftreated animal cells (from an animal suspected of infection withChlamydia) is placed on the automated stage.

As illustrated in FIG. 2, a slide is loaded (200), (preferably using anautomated slide loader.

The program moves the automated stage to a preset starting point, e.g.,on a corner of the slide.

Optionally, the x-y position of the stage at the starting point isrecorded.

An optical field is selected and the microscopic image is acquired andtransferred to the computer as an RUB image (201).

The Red component of the RGB image is transferred to a new monochromegrey-level image and clipped for pixel values of less than 50 to cutdown signal noise (202).

The grey-level image is transformed to a binary image, a black and whiteimage in which pixels with corresponding pixels in the Luminance imagehaving grey-level values lower than the cut off point are set to 255(white) (203).

Noise particles are removed and a hole filling function is applied tothe binary image (204). In an illustrative embodiment, (see infraSection 6) a commercially available MATROX function is used to fill theholes in the identified images. The area of each image is measured andall images having an area of expected for a monocyte or mononuclearphagocyte, i.e., 1000 pixels or greater are selected as representativeof mononuclear phagocytes. Cell images that have an area less than 1000pixels are excluded from further processing (205).

The area, in pixels, of the cell images, i.e., mononuclear phagocytes,is recorded and saved for further processing (206). Optionally, the XYlocation of mononuclear phagocytes is recorded for later analysis and/orprocessing.

The original Red component from the original RGB image is transferred toa binary image, so that pixels having grey-level values less than 200are set to 0 while all the rest are set to 255 (207 a).

The original Green component is transferred to grey level image (207 b).

All pixels that have a value of M and any grey-level value in the Greencomponent of the original RUB image equal to N form a new grey-levelimage (208). In a specific illustrative embodiment exemplified infra inSection 6 when a first signal indicative of a monocyte is generatedusing counter stain with 4′-6-Diamidino-2-phenylindole is detected and asecond signal indicative of Chlamydia is detected using fluorophoreconjugated ChlamydiaCel monoclonal antibody available from (CelLabs,Sydney, Australia) is used, M is set at 255 and N is set at 0.

This new grey-level image is transformed into a binary image wherepixels that have grey-level values less than 100 are set 0 and the restof the pixels are set to 255 (209).

If the level is 255, the monocyte is counted and recorded as anChlamydia-infected monocyte (212); if not, the monocyte is recorded asnot infected (211).

The extent of infection of individual Chlamydia infected monocytes canbe determined:

An opening filter is applied to remove small noise particles from thebinary image and a hole filling function is applied (214)

The total area of the remaining cell images, which represents the totalarea of Chlamydia organisms within the cell perimeter is recorded (215)

Percent area of each mononuclear phagocyte that is occupied by Chlamydiaorganisms can be calculated (216).

As illustrated in FIG. 2, according to one mode of this embodiment ofthe invention, detection of Chlamydia infected monocytes can simply berecorded for each image (210-212) or the process can be repeated for anumber of optical fields and a number of images can be accessed (218) upto an including the entire surface of a slide (218-217). Simpledetection of infected monocytes provides useful information fordiagnosis and/or prognosis as described in detail in Section 5.2, infra.In addition, the number of infected monocytes can be determined toprovide even more useful information as described in Section 5.2, infra.

Alternatively, as further illustrated in FIG. 2, the percent area of amonocyte occupied by Chlamydia is calculated (213-216). This providesquantitative information regarding extent of infection useful fordiagnosis and/or prognosis as described in detail in Section 5.2, infra.

FIG. 3 schematically illustrates an alternative mode of this embodimentof the invention. As illustrated in FIG. 3, according to an alternativeof this mode of the embodiment of the invention, the number andpositions of all monocytes on a slide are determined. In this mode ofthis embodiment of the present invention, Chlamydia infection ofmonocytes can be detected or detected and quantified using the stepsillustrated in FIG. 2 either after recordation of each monocyte image(see FIG. 3, 307) or after all monocyte images have been recorded (seeFIG. 3, 309). This provides information useful as described in Section5.2, infra.

As would be understood by those skilled in the art, the parameters toset levels of image detection for animal cells and for infectious agentswill depend upon the specific animal cells and infectious agents to bedetected as well as upon the specific first and second signals used todetect the cells and infectious agents. Such parameters can bedetermined by those skilled in the art using known methods and theinformation provided herein.

5.1.2. METHODS FOR DETECTION OR DETECTION AND QUANTIFICATION OF AFREE-FLOATING AGENT

According to another embodiment, an infectious agent is detected orpreferably detected and quantified by computer controlled image analysisof a free floating infectious agent in a sample of a body fluid ortissue from an animal. In this embodiment, computer controlled imageanalysis is conducted on a sample of body fluid or tissue treated toprovide at least one signal specific to the infectious agent of interestwhich can be detected and quantified. As would be understood by thoseskilled in the art, the same signals specific for an infectious agentdescribed in Section 5.1.1., supra, are suitable for use in thisembodiment of the invention.

A slide containing, for example, a blood smear from a patient suspectedof infection with Chlamydia is properly immunostained, for example,using the following steps: blood smears are washed withphosphate-buffered saline, fixed with methanol and stained directly withChlamydia genus-specific FITC-conjugated monoclonal antibody obtainede.g. from (Kallestad Diagnostics, Chaska, Minn.).

Images are captured at 1000.times. total magnification. The PC (see FIG.1, 106) software program compiled in MICROSOFT C++ using the MATROXIMAGING LIBRARY (MIL). MIL is designed by MATROX to control theoperation of the frame grabber and the processing of images captured byframe grabber and subsequently stored in PC as disk files. It comprisesa number of image processing specialized routines particularly suitablefor performing such image processing task as filtering, object selectionand various measurement functions. The program prompts and measurementresults are shown on the computer monitor (see FIG. 1, 108).

FIG. 4 schematically illustrates this embodiment of the method of theinvention, using Chlamydia as the infectious agent, merely forillustrative purposes and ease of explanation.

As illustrated in FIG. 4, detection or detection and quantification offree floating Chlamydia in a blood sample on a microscope slide isachieved as follows:

An optical field is selected and the microscopic image is acquired andtransferred to the computer as an ROB image (401);

The image is transferred in the HLS domain (402);

The Hue component of the HLS image is transformed to a new monochromegrey-level image (403);

A “binary” image is formed: this is a black and white image in whichpixels with pixels having values between 10 and 25 in the Hue image setto 255 (white) and the rest being set to 0 black (404);

An “open” filter is applied: Opening is a successive application of an“erosion” filter followed by a “dilation” filter. It allows for theremoval of small noise particles from the binary image. A hole fillingfunction is applied. (405);

The area of the blobs is measured and all blobs that have an area ofless than 30 pixels and more than 70 are excluded from furtherprocessing. The remaining blobs represent identified Chlamydia particlesin the blood sample (406);

The pixel number (area) of these blobs (cells) is recorded and saved forfurther processing (407).

5.2. DISEASES, DISORDERS AND APPLICATIONS

The information provided by the computer controlled image analysismethods of the present invention is useful, preferably in combinationwith other information relating to the genetic/hereditary, physicaland/or physiological state of a human or non-human patient, in diagnosisor prognosis of a disease or disorder associated with infection of thehuman or non-human patient by an infectious agent. Prognosis encompassesdetermination of the susceptibility to disease onset or progression,spread or recurrence, of extent of disease or of whether a treatment apatient is undergoing is effective in ameliorating, preventing or curinga disease or disorder from which the patient is suffering. Theinformation provided is useful for diagnosis or prognosis of diseasesand disorders associated with a variety of infectious agents, including,but not limited to, prions, bacteria, mycoplasma, rickettsia,spirochetes, fungi, protozoal parasites, viruses, etc.

In certain embodiments, the methods provide information useful andsufficient to diagnose a disease or disorder caused by an infectiousagent. As an illustrative example, the methods of the invention are usedto detect or detect and quantitate the presence of Chlamydia pneumoniaein a body fluid or tissue sample to diagnose the presence of pneumoniacaused by said infectious agent. As another illustrative example themethods of the invention are used to detect and quantitate the presenceof Borrelia in a body fluid or tissue sample to diagnose the presence ofLyme disease caused by said infectious agent.

Thus, in certain embodiments, simple detection of the presence of aninfectious agent either in an animal cell or free floating in a bodyfluid or tissue provides sufficient information to diagnose a diseasecaused by the infectious agent. In other embodiments, quantitativeinformation regarding the extent or presence of an infectious agent,e.g. a virus is useful for prognosis of a disease cause by theinfectious agent.

in yet other embodiments, the quantitative information regarding aninfectious agent provided by the methods of the invention is used todetermine therapeutic efficacy of a treatment which a patient infectedwith an infectious agent is undergoing.

In an important specific embodiment, the invention provides is a uniquemethod to detect mononuclear phagocytes infected with Chlamydia,Helicobacter or a limes virus to provide information useful fordiagnosis or prognosis of vascular disease, such as atherosclerosis orarteriolosclerosis or coronary disease, such as congestive heart failureor susceptibility to myocardial infarction. Rather than identifyingvascular disease, such as atherosclerosis, with invasive procedures byviewing the manifestations of fatty streaks, intermediate lesions, orfibrous plaques, this embodiment of the present invention providesinformation useful for determination of atherosclerosis orsusceptibility to same using a minimally-invasive procedure by analysisof a blood sample, e.g., a peripheral blood sample. Determination of achange, especially an increase in the number of infected monocytes isimportant for a prognosis of disease since published epidemiologicalstudies of vascular and coronary heart disease show at least a two-foldor larger odds ratio with the presence of infected monocytes. Some showincreasing odds ratios with increasing antibody titres.

In another specific embodiment, the invention provides a unique methodto detect and preferably quantitate the presence of an infectious agentsuch as Chlamydia in blood, spinal fluid or other body fluid. Thisinformation is particularly useful in determining the etiology ofcertain diseases or disorders of the central nervous system.

6. EXEMPLARY EMBODIMENT

The following non-limiting example illustrates the detection ofChlamydia pneumoniae infected mononuclear phagocytes to provideinformation useful for prognosticating atherosclerosis susceptibility.In this example, a peripheral blood sample is obtained from a patientsuffering from at least one of the signs and symptoms ofatherosclerosis. Using the automated image analysis method of thepresent invention, quantitative determination is made of the extent ofChlamydia infection of the patient's monocytes. Based on the informationprovided, a determination can be made regarding whether or not a courseof treatment with antibiotic(s) effective against Chlamydia would beuseful for ameliorating or preventing progression of at least onesymptom or sign of atherosclerosis.

6.1. SMEAR PREPARATION

A non-coagulated whole blood sample is subjected to centrifugationthrough a Ficoll-Histopaque density gradient. The fraction containingmononuclear phagocytes is collected and washed with phosphate-bufferedsaline. Smears are prepared from aliquots of cells on glass microscopeslides.

6.1.1. CELL FIXATION

Smears are fixed in methanol for 5-10 minutes. Alternatively, smears maybe fixed in ice-cold 0.05% glutaraldehyde for 10-30 minutes at roomtemperature. The smears are then washed with phosphate-buffered salinecontaining 0.1% bovine serum

6.1.2. CELL STAINING

Staining for C. pneumoniae infected mononuclear phagocytes entailsincubating the cells with fluorophore-conjugated genus-specificanti-Chlamydial monoclonal antibody for 15-60 minutes at roomtemperature in the dark. For example, ChlamydiaCel (available fromCelLabs, Sydney, Australia) can be used. The smear then iscounterstained with 4′,6-Diamidino-2-phenylindole (“DAPI”) for 5 minutesat room temperature. The smear is washed with phosphate-buffered salineand allowed to air dry.

6.2. AUTOMATED SMEAR ANALYSIS

C. pneumoniae infected mononuclear phagocytes are identified to provideinform useful to prognosticate atherosclerosis susceptibility. Automatedimage analysis of the smear is conducted as described in Section 5.1.1,supra. A description of the detailed method used in the exemplaryembodiment follows.

6.2.1. METHOD

The PC executes a smear analysis software program compiled in MICROSOFTC++ using the MATROX IMAGING LIBRARY (MIL). MIL is a software library offunctions, including those which control the operation of the framegrabber and which process images captured by the frame grabber forsubsequent storage in PC as disk files. MIL comprises a number ofspecialized image processing routines particularly suitable forperforming such image processing tasks as filtering, object selectionand various measurement functions. The smear analysis software programruns as a WINDOWS 95 application. The program prompts and measurementresults are shown on the computer monitor, while the images acquiredthrough the imaging hardware are displayed on the dedicated imagingmonitor.

6.2.2. DETECTION OF THE SIGNAL

The Chlamydia infected mononuclear phagocyte detection algorithm isillustrated in the flow charts of FIGS. 2 and 3, where possible cellpositions are identified at 1000.times. total magnification. The100.times. objective is selected and the search for C. pneumoniaeinfected mononuclear phagocyte is conducted:

An optical field is selected and the microscopic image is acquired andtransferred to the computer as an RGB image (201).

The Red component of the RGB image is transferred to a new monochromegrey-level image and clipped for pixel values of less than 50 to cutdown signal noise (202).

The grey-level image is transformed to a binary image, a black and whiteimage in which pixels with corresponding pixels in the Luminance imagehaving grey-level values lower than the cut off point are set to 255(white) (203).

An opening filter, successive applications of an erosion filter followedby a dilation filter, is applied for the removal of small noiseparticles from the binary image (204).

Application of a hole filling function fills the holes in the identifiedimages (204). In this illustrative example, the hole filling function iscommercially available as a MATROX function from Matrox (Montreal,Canada).

The area of each image is measured and all images having an area of 1000pixels or greater are representative of mononuclear phagocytes. Cellimages that have an area less than 1000 pixels are excluded from furtherprocessing (205).

The area, in pixels, of the cell images, i.e., mononuclear phagocytes,is recorded and saved for further processing (206).

The original Red component from the original RGB image is transferred toa binary image, so that pixels having grey-level values less than 200are set to 0 while all the rest are set to 255 (207 a).

The original Green component is transferred to grey level image (207 b).

All pixels that have a value of 255 and any grey-level value in theGreen component of the original RGB image form a new grey-level image(208).

This new grey-level image is transformed into a binary image wherepixels that have grey-level values less than 100 are set 0 and the restof the pixels are set to 255 (209).

The monocyte can be counted and recorded as an uninfected orChlamydia-infected monocyte (210-212), or

An opening filter is applied to remove small noise particles from thebinary image (214).

The total area of the remaining cell images, which represents the totalarea of Chlamydia organisms with in the cell perimeter, is recorded.(215).

Percent area of each mononuclear phagocyte that is occupied by Chlamydiaorganisms can be calculated (216).

As further illustrated in FIG. 2, all optical fields on a slide can beassessed (FIG. 2, 217-118). The algorithm is repeated for additionalfields of the smear according to the flow chart of FIG. 3. AfterChlamydia infected mononuclear phagocytes are identified and quantified,the quantitative information is used, in combination with other relevantinformation regarding the physical and physiological state of thepatient's vascular system to determine whether a course of antibiotictreatment is indicated for the patient.

Computer and image processing technologies are constantly changing.Newer technologies which meet the needs of the above-described methodsand apparatus, while not specifically described here, are clearlycontemplated as within the invention. For example, certain conventionalpixel and image file formats are mentioned above, but others may also beused. Image files may be compressed using JPEG or GIF techniques nowknown in the art or other techniques yet to be developed. Other colorspaces may also be used, as desired by the skilled artisan, particularlywhen detection of a sought-after characteristic is enhanced thereby.

The present invention has now been described in connection with a numberof particular embodiments thereof. Additional variations should now beevident to those skilled in the art, and are contemplated as fallingwithin the scope of the invention.

All references cited herein are incorporated herein by reference intheir entirety.

What is claimed:
 1. A computer software product, comprising acomputer-readable storage medium having fixed therein a sequence ofinstructions which, when executed by a computer, direct the performanceof a method which comprises: a) acquiring a microscope image of anoptical field of a substrate having fixed thereon a monolayer of animalcells which either produce or are treated to produce a first signalspecific to an animal cell of interest and treated to produce a secondsignal specific to an infectious agent of interest, if present, andtransferring the image to an ROB image; b) transferring the Redcomponent of the RGB image to a new monochrome grey image; c)transforming the grey level image to a binary image using a cut offpoint set to a value indicative of the expected size of the animal cellsof interest; d) operating on the binary image to remove noise and fillholes; e) measuring size of the image of step d), selecting andrecording areas representative of the animal cells of interest; f)transferring the Red component of the original ROB image to a secondbinary image using an expected value indicative of the infectious agentof interest; g) transferring the Green component of the original RGBimage to grey level; h) forming a new grey level image using all pixelshaving a value equal to a set value M and any grey level value in theGreen component in the original RGB image equal to a set value N; i)transforming the new grey level images to a third binary image; j)operating on the third binary image to remove noise and fill holes; k)recording the area of the third binary image after step j); and l)determining whether or not, each identified animal cell is occupied byan infectious agent of interest.
 2. The computer software product inaccordance with claim 1, wherein the method further comprisescalculating the extent of each animal cell occupied by said infectiousagent.
 3. The computer software product in accordance with claim 1,further comprising a computer-readable storage medium having fixedtherein a sequence of instructions which, when executed by a computer,direct the performance of a method which comprises: a) acquiring amicroscope image of an optical field of a substrate having fixed thereona sample of a body fluid or tissue from an animal patient suspected ofcontaining an infectious agent of interest, which sample either producesor is treated to produce a first signal specific to an infectious agentof interest, if present, and transferring the image to an RGB image; b)transferring the RGB image to an HLS image and transforming the HISimage to a new monochrome grey level image; c) transforming the greylevel image to a binary image; d) operating on the binary image using atleast one filter to identify blobs; and e) selecting blobs based on theexpected size of area of an infectious agent to determining whether ornot an infectious agent of interest is present in the sample.